Induction of Glo1 expression by tRES and HESP Induction of Glo1 expression by tRES and HESP. GLO1-ARE transcriptional response reporter assay. Induction of Glo1 expression by tRES and HESP. GLO1-ARE transcriptional response reporter assay. Data of normalized responses for varied bioactive concentrations were fitted by nonlinear regression to the equation E = Emax × [bioactive]n/(EC50n + [bioactive]n), solving for Emax, EC50, and n (Hill coefficient). A: Concentration-response curve for tRES. Data are mean ± SD (n = 3) for 5 concentrations. Nonlinear regression (red curve): E (%) = 100 × [tRES]3.92/(2.523.92 + [tRES]3.92). B: Concentration-response curve for HESP. Data are mean ± SD (n = 3–8) for 6 concentrations. Nonlinear regression (blue curve): E (%) = 24.4 × [HESP]2.01/(0.592.01 + [HESP]2.01). C: Concentration-response curve for tRES in the presence of 5.0 μmol/L HESP. Data are mean ± SD (n = 3–6) for 5 concentrations. Nonlinear regression, green curve: E (%) = (83.4 ×[tRES]1.36/(1.461.36 + [tRES]1.36)) + 11.6; green curve: tRES+ 5.0 μmol/L HESP; red dotted curve: tRES only (as for A). D: Expansion of C, also showing the response for 5.0 μmol/L HESP (blue dashed line). E–H: Evaluation of the effect of tRES and HESP individually and in combination on the growth and viability of BJ fibroblasts (E and F) and HAECs (G and H) in vitro. For E and G, cells were incubated with 0.625–40 μmol/L tRES and with (green bars) or without (red bars) 5.0 μmol/L HESP. For F and H, cells were incubated with 0.625–40 μmol/L HESP and with (green bars) or without (blue bars) 5.0 μmol/L tRES. I–K: Validation of induction of Glo1 expression by 5.0 μmol/L tRES and HESP, individually and combined. Cell type: HAECs (I), BJ fibroblasts (J), and HepG2 (K) cells. Panels (from left to right): GLO1 mRNA change with 5.0 μmol/L tRES (red line), 5.0 μmol/L HESP (blue line), and 5.0 μmol/L tRES and HESP (green line). Bar chart: Glo1 protein (16 h posttreatment) plus 5.0 μmol/L tRES (red bar) and 5.0 μmol/L HESP (blue bar). Data are mean ± SD (n = 3) for E–K. Significance: *P < 0.05, **P < 0.01, and ***P < 0.001. HAEC cells were grown in proprietary large-vessel endothelial cell basal media supplemented with large-vessel endothelial cell growth supplement (containing hydrocortisone, human epidermal growth factor, and human fibroblast growth factor with heparin and in 2% [v/v] FBS), 25 µg/mL gentamicin, and 50 ng/mL amphotericin B. They were cultured in minimum essential medium with 10% FCS and 2 mmol/L glutamine under an atmosphere of 5% CO2 in air, 100% humidity, and 37°C. Mingzhan Xue et al. Diabetes 2016;65:2282-2294 ©2016 by American Diabetes Association